The development of new biophysical techniques is of paramount importance to the advancement of science. This proposal aims to advance existing spectroscopic imaging techniques to elucidate the understanding of cellular membranes and to further this understanding by developing both new model systems and techniques with unprecedented chemical and spatial resolution for application to biological systems. A Raman near fleld scanning optical microscope (NSOM) was developed during the K99 phase that will enable vibrational spectroscopic imaging with nanoscale spatial resolution. Vibrational spectroscopic imaging offers the opportunity to spatially resolve biological systems solely on the basis ofthe molecules present. This enables the study of the dynamical and organizational characteristics of cells and tissue without the need for external labels, which can disrupt the behavior of the system. This proposal aims to further develop and apply these new techniques to elucidate further the characteristics of biomolecules located within cellular membranes. Specifically, model systems will be utilized to explore the biophysical insights gained from this new methodology. Planar supported bilayers will be used as a model system for domain formation motifs, particularly the chemical composition and interactions that are found in lipid domains. l\/Iore sophisticated model systems, such as confined vesicles, will be developed that will facilitate the ultimate goal of observing and understanding membrane heterogeneity and its role in cellular membrane processes such as signaling, and transport across the cell membrane. Ultimately, this proposal will utilize the established instrumentation and advance the methodology to gain insight into intact cell membranes. While domains are commonly ambiguously implicated in cell membrane processes related to pathogen infection, transport, and protein receptor activity, this research will demonstrate and clarify these molecular interactions at levels previously unattainable.